Gel electrophoresis has become an increasingly indispensable tool in biotechnology and related fields. The ability to separate molecules by means of size, shape and charge has added numerous opportunities to identify specific compounds, determine purity, and allow for isolation of a compound in a relatively pure form. By being able to change the conditions under which one carries out the electrophoresis, one can determine many characteristics of the compounds in the sample.
A variety of analytical techniques are predicated on the use of gel electrophoresis in an efficient and convenient way. Gel electrophoresis may be used to identify a compound, where the components of a complex mixture are first separated and then subsequently identified by using markers such as antibodies, or the like. Gel electrophoresis may also be used in the determination of the molecular weights of macromolecules, such as proteins.
In many analytical techniques based on an initial separation of the sample components, further analysis of the separated components requires a transfer or blotting step. In blotting steps, the separated components, e.g. proteins, ribonucleic acids and deoxyribonucleic acids are transferred to an alternative substrate, such as a membrane matrix, derivitized paper, or the like. Exemplary blotting methods include Southern, northern and western blotting. The advantages of including a blotting step in a particular analytical technique include increased accessibility of the electrophoretically separated macromolecules to the reagents of a particular assay, as compared to macromolecules that remain in the gel. Further, smaller amounts of reagent are required and shorter incubation times are often observed for blotted macromolecules as compared with macromolecules that remain in the gel matrix during an assay. Thus, most assay protocols for electrophoretically separated components still require that the separated components be transferred from the gel to a substrate prior to the addition of assay reagents.
Despite the benefits of blotting as an analytical technique, there are disadvantages to the inclusion of a blotting step. One disadvantage in blotting is the loss of components during transfer from the gel to the substrate. Another disadvantage of blotting is the inefficient transfer of larger macromolecules. Although various methods have been devised to compensate for inefficient transfer e.g. treatment with Pronase to cause limited digestion of large proteins in the gel, these methods denature the transferred proteins. Such denaturation can adversely affect the activity of an enzyme. Further, the inclusion of a blotting step adds complexity to the overall analytical technique. Upon inclusion of a blotting step, the possibilities for the introduction of error into the overall protocol increases. Thus, there are significant disadvantages to using a transfer step in an analytical process.
Despite the numerous disadvantages associated with blotting, the number of assays that can be conducted on electrophoretically separated components directly on a gel remains limited. Therefore, there is a continuing need for the development of new methods of electrophoresis that allow for a broader array of assays to be conducted in the gel itself, thereby avoiding the need for a blotting step, as well as the concomitant disadvantages associated with blotting.